ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2.

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Murine Alox8 versus the human ALOX15B ortholog: differences and similarities.

Human arachidonate 15-lipoxygenase type B is a lipoxygenase that catalyzes the peroxidation of arachidonic acid at carbon-15. The corresponding murine ortholog however has 8-lipoxygenase activity. Both enzymes oxygenate polyunsaturated fatty acids in S-chirality with singular reaction specificity, although they generate a different product pattern. Furthermore, while both enzymes utilize both esterified fatty acids and fatty acid hydro(pero)xides as substrates, they differ with respect to the orientation of the fatty acid in their substrate-binding pocket. While ALOX15B accepts the fatty acid "tail-first," Alox8 oxygenates the free fatty acid with its "head-first." These differences in substrate orientation and thus in regio- and stereospecificity are thought to be determined by distinct amino acid residues. Towards their biological function, both enzymes share a commonality in regulating cholesterol homeostasis in macrophages, and Alox8 knockdown is associated with reduced atherosclerosis in mice. Additional roles have been linked to lung inflammation along with tumor suppressor activity. This review focuses on the current knowledge of the enzymatic activity of human ALOX15B and murine Alox8, along with their association with diseases.

Cholesterol homeostasis in hair follicle keratinocytes is disrupted by impaired ABCA5 activity.

The importance of cholesterol in hair follicle biology is underscored by its links to the pathogenesis of alopecias and hair growth disorders. Reports have associated defects in ABCA5, a membrane transporter, with altered keratinocyte cholesterol distribution in individuals with a form of congenital hypertrichosis, yet the biological basis for this defect in hair growth remains unknown. This study aimed to determine the impact of altered ABCA5 activity on hair follicle keratinocyte behaviour. Primary keratinocytes isolated from the outer root sheath of plucked human hair follicles were utilised as a relevant cell model. Following exogenous cholesterol loading, an increase in ABCA5 co-localisation to intracellular organelles was seen. Knockdown of ABCA5 revealed a dysregulation in cholesterol homeostasis, with LXR agonism leading to partial restoration of the homeostatic response. Filipin staining and live BODIPY cholesterol immunofluorescence microscopy revealed a reduction in endo-lysosomal cholesterol following ABCA5 knockdown. Analysis of oxysterols showed a significant increase in the fold change of 25-hydroxycholesterol and 7-ß-hydroxycholesterol following cholesterol loading in ORS keratinocytes, after ABCA5 knockdown. These data suggest a role for ABCA5 in the intracellular compartmentalisation of free cholesterol in primary hair follicle keratinocytes. The loss of normal homeostatic response, following the delivery of excess cholesterol after ABCA5 knockdown, suggests an impact on LXR-mediated transcriptional activity. The loss of ABCA5 in the hair follicle could lead to impaired endo-lysosomal cholesterol transport, impacting pathways known to influence hair growth. This avenue warrants further investigation.

SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.

Arachidonate 15-lipoxygenase type B: Regulation, function, and its role in pathophysiology.

As a lipoxygenase (LOX), arachidonate 15-lipoxygenase type B (ALOX15B) peroxidizes polyenoic fatty acids (PUFAs) including arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and linoleic acid (LA) to their corresponding fatty acid hydroperoxides. Distinctive to ALOX15B, fatty acid oxygenation occurs with positional specificity, catalyzed by the non-heme iron containing active site, and in addition to free PUFAs, membrane-esterified fatty acids serve as substrates for ALOX15B. Like other LOX enzymes, ALOX15B is linked to the formation of specialized pro-resolving lipid mediators (SPMs), and altered expression is apparent in various inflammatory diseases such as asthma, psoriasis, and atherosclerosis. In primary human macrophages, ALOX15B expression is associated with cellular cholesterol homeostasis and is induced by hypoxia. Like in inflammation, the role of ALOX15B in cancer is inconclusive. In prostate and breast carcinomas, ALOX15B is attributed a tumor-suppressive role, whereas in colorectal cancer, ALOX15B expression is associated with a poorer prognosis. As the biological function of ALOX15B remains an open question, this review aims to provide a comprehensive overview of the current state of research related to ALOX15B.

Efferocytosis potentiates the expression of arachidonate 15-lipoxygenase (ALOX15) in alternatively activated human macrophages through LXR activation.

Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann-Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRß in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs.